Preparation of organotypic hippocampal slice cultures: interface method
This protocol describes a method for making and culturing rat hippocampal organotypic slices on membrane inserts. Supplementary videos are included to demonstrate visually the different steps of the procedure. Cultured hippocampal slices has been increasingly used as a model for synaptic studies of the brain as they allow examination of mid to long term manipulations in a preparation where the gross cytoarchitecture of the hippocampus is preserved. Combining techniques such as molecular biology, electrophysiology and immunohistochemistry to study physiological or pathological processes can easily be applied to organotypic slices. The technique described here can be used to make organotypic slices from other parts of the brain, other rodent species and from a range of ages. This protocol can be completed in 3 h.
Comments
This protocol is a god send, especially with the videos, I have two questions:
1) To visualize the organotypic slices for electrophysiology, does one need an IR-DIC microscope? Or are they thin enough that a standard upright phase contrast microscope will do?
2) Would one be able to do the dissection and slicing outside of the sterile flow cabinet in a semi-sterile environment? So that one can use a neuroprotectant sucrose aCSF during slicing, or use a microtome that is too large to move in and out of a flow cabinet? The simply wash the brain slices several times with sterile HEPES-EBSS before placing them on the confetti?
Posted by: Bill Connelly | July 19, 2007 04:43 AM
Thank you for your comment. A quick answer to your questions:
1) The IR-DIC microscope is optimal for visualisation and electrophysiology of organotypic slices, especially within the first 10 days in vitro. From DIV14 onwards, when the slice has thinned down to 3-4 layers of cells, one with expertise in patching hippocampal slices could try doing electrophysiology using a standard upright phase contrast microscope (but expecting the number of successful patches being less compared with IR-DIC). CA1 cell bodies would be quite easily recognisable at this stage, while other smaller cells could represent a problem, for which IR-DIC microscope is better indicated.
2) We recommend the use of a sterile flow cabinet for dissection and slicing. The culture medium would quickly grow every contaminant, even if present in small amount. We indeed had occasional contamination albeit the use of strict sterile measures. The semi-sterile environment described could possibly be used when organotypic slices are going to be studied at a very early stage, i.e. DIV1 or maximum DIV2, when contamination is still contained and some viable cells could be present.
For future comments/communications with the corresponding author, please use the following address: a.desimoni@nhs.net
Posted by: Anna De Simoni | October 17, 2007 03:31 PM